This is a competitive revision of parent grant GM078097, Mutational Studies of Processive Myosin Motors. It seeks to expand the scope of the original application by proposing experiments with motor proteins in cultured mammalian cells, in contrast to the parent application which purely involved in vitro characterization. Moreover, it fulfills both the role of job creation (a new postdoctoral associate will be hired) as well as accelerating the tempo of research by this input of manpower. The first aim seeks to establish what features of myosin V are essential for it to effectively maneuver through the meshwork that constitutes the actin cortex in a living cell. The importance of duty cycle of the motor and motor number per cargo for myosin V based cargo-transport will be determined in COS-7 cells. Secondly, we will investigate how the handoff of cargo, from kinesin-based movement on microtubules to myosin Vbased transport on actin, is facilitated within the cell. Kinesin and myosin V will be attached to the same cargo to follow transport from the cell center out to the periphery. The impact of this work will be to establish the extent to which the basic properties we associate with myosin V and kinesin from in vitro studies are also operative within the cell. A number of human diseases result from disruption of cargo transport along actin or microtubules, or from disease proteins acting as cargo for these transport proteins. A better understanding of the mechanism by which molecular motors move along their track in the cell is critical to further understand the basis of these diseases. PUBLIC HEALTH RELEVANCE: A number of human diseases result from disruption of cargo transport along actin or microtubules, or from disease proteins acting as cargo for these transport proteins. A better understanding of the mechanism by which molecular motors move along their track in the cell is critical to further understand the basis of these diseases.